How Is Arachidonic Acid Metabolism in the Uterus Connected with the Immune Status of Red Deer Females (Cervus elaphus L.) in Different Reproductive Stages?

Reproductive and condition parameters' dependency on immune status in seasonally reproducing ruminants such as red deer have not been outlined to date. We determined T and B blood lymphocytes; the concentration of IgG, cAMP, haptoglobulin, and 6-keto-PGF1α in blood plasma; and the mRNA and protein expression of PG endoperoxide synthase 2, 5-lipoxygenase, PGE2 synthase (PGES), PGF2α synthase (PGFS), PGI2 synthase (PGIS), leukotriene (LT)A4 hydrolase, and LTC4 synthase (LTC4S) in the uterine endo- and myometrium, on the 4th (N = 7) and 13th (N = 8) days of the estrous cycle, in anestrus (N = 6) and pregnancy (N = 8) in hinds. An increase in CD4+ T regulatory lymphocyte percentage during the estrous cycle and anestrus compared with pregnancy was recorded; the opposite effect was observed for CD21+ B cells (p < 0.05). cAMP and haptoglobin concentration were elevated during the cycle, as was IgG on the fourth day of the cycle, whereas 6-keto-PGF1α concentration was the highest in pregnancy, and the nearest in anestrus similarly were LTC4S, PGES, PGFS, and PGIS protein expression in the endometrium (p < 0.05). We showed an interaction between the immune system activation and AA-metabolite production in the uterus throughout different reproductive stages. IgG, cAMP, haptoglobin, and 6-keto-PGF1α concentrations are valuable candidates for markers of reproductive status in hinds. The results help expand our knowledge of the mechanisms underlying seasonal reproduction in ruminants.

The Quality and Fertilizing Potential of Red Deer (Cervus elaphus L.) Epididymal Spermatozoa Stored in a Liquid State.

The aim of this study was to assess the quality and fertilizing potential of red deer epididymal spermatozoa stored in a liquid state for up to 11 days (D11). In Experiment 1, sperm quality was determined. In Experiment 2, the efficiency of in vitro fertilization (IVF) and artificial insemination (AI) of stored sperm were evaluated. An analysis of sperm quality on D5 of storage revealed a decrease (p < 0.05) in motility and morphology, and a higher proportion of apoptotic spermatozoa. On D1, D7 and D10, the total motility of sperm for IVF and AI was determined to be 82.6%, 71.0% and 64.8%, respectively. The results of IVF and AI demonstrated that the fertilizing potential of spermatozoa differs between days of storage. The percentage of blastocysts was higher when oocytes were fertilized on D1 (17.4 %) compared to D7 (8.5%) and D10 sperm (10.5%). Differences were noted in the pregnancy rates of inseminated hinds. The insemination with D1, D7 and D10 sperm led to live births (33% from D7 and D10). The results indicate that the quality of red deer epididymal spermatozoa remains satisfactory during ten days of storage in a liquid state, and that these spermatozoa maintain their fertility potential.

Regulation of uterine function during estrous cycle, anestrus phase and pregnancy by steroids in red deer (Cervus elaphus L.).

Steroid synthesis and production in ruminant uterus is not obvious, especially in seasonally reproduced. We compared steroid production by investigating enzymes involved in red deer uterine steroid metabolism in reproductive seasons. Blood and uteri (endometrium and myometrium) were collected post mortem from hinds on 4th day (N = 8), 13th day of the cycle (N = 8), anestrus (N = 8) and pregnancy (N = 8). The expression of cytochrome P450 aromatase (P450), 3 -beta-hydroxysteroid dehydrogenase (3ß-HSD), 17 -beta-hydroxysteroid dehydrogenase (17ß-HSD), aldo-keto reductase family 1 C1 (AKR1C1), estrogen receptor alpha (ERα), and progesterone receptors (PRs), were analyzed using real-time-PCR and Western Blotting. Plasma samples were assayed for 17-beta-estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T4) concentrations by EIA. Hinds at the beginning of the estrous cycle, mainly in endometrium, were characterized by a high mRNA expression of 3ß-HSD, AKR1C1, PRs and ERα, contrary to the expression in myometrium during pregnancy (P < 0.05). For P4, E2, and FSH, concentration was the highest during the 13th day of the estrous cycle (P < 0.05). Uterine steroid production and output in hinds as a representative seasonally reproduced ruminant occurred mainly during the estrous cycle and sustained in anestrus.

Artificial Reproductive Technology (ART) Applied to Female Cervids Adapted from Domestic Ruminants.

There are about 150 Cervidae species on the IUCN Red List of Threatened Species. Only a small part is counted among farm animals, and most of them are free roaming. The universality and large numbers of representatives of cervids such as red deer (Cervus elaphus) and roe deer (Capreolus capreolus) may predispose these species to be used as models for research on reintroduction or assisted reproduction of deer at risk of extinction. We outlined the historical fluctuation of cervids in Europe and the process of domestication, which led to breeding management. Consequently, the reproductive techniques used in domestic ruminants were adapted for use in female deer which we reviewed based on our results and other available results. We focused on stress susceptibility in cervids depending on habitat and antropopression and proposed copeptin as a novel diagnostic parameter suitable for stress determination. Some reproductive biotechniques have been adopted for female cervids with satisfactory results, e.g., in vitro fertilization, while others still require methodological refinement, e.g., cryopreservation of oocytes and embryos.

The Effectiveness of Pharmacological Synchronization of the Estrous Cycle in Hinds (Cervus elaphus L.): A Pilot Field Trial.

The aim was to estimate the effective pharmacological method of the estrous cycle synchronization by checking the effects of synchronization by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA and artificial insemination. The experiment was performed at the red deer farm in Rudzie (North-East Poland; 3 year's old). The herd (N = 14) was kept away from bulls and was divided in two groups of seven animals. In the Group I, CIDR insert (0.3 g of P4) was applicated intravaginally for 12 days; a second insert replaced the first one for the next 12 days, and next 200 IU of equine chorionic gonadotropin (eCG) was injected intramuscularly (Folligon). Estrus was expected 48 h after eCG injection. In the Group II, Chronogest sponge (20 mg of flugestone acetate) was applicated intravaginally and after 7 days replaced with second chronogest sponge for 7 days. After removing the sponge, on the same day eCG was injected and estrus was expected after 48 h. Artificial insemination was provided with frozen-thawed semen twice: 12 and 24 h after expected estrus. The peripheral blood from the jugular vein was collected each time when the inserts or sponge were applicated and 40 days after insemination. The concentration of P4 and E2 in plasma was measured by RIA. The effectiveness of insemination was monitored by pregnancy-associated glycoproteins determination and observed by the number of calves born. Two pregnancies were confirmed in Group I and five in Group II based on PAG concentration. One newborn was observed in Group I and five in Group II. Both methods of synchronization are effective in hinds based on the received profile of steroids. Although the sponge shape in case of chronogest is better comparing with CIDR, which was not completely deposited in the vagina of hind, potentially leads to bacteria inflammation, and it disturbs the rightful endocrine regulation. Moreover, pregnancy rate and hormone responsiveness were better in Group II.

Antioxidative Potential of Red Deer Embryos Depends on Reproductive Stage of Hind as A Oocyte Donor.

The aim was to compare the blastocyst stages of red deer embryos in respect of in vitro fertilization (IVF) efficiency, morphology, apoptotic and proliferative abilities, and antioxidative potential according to the reproductive status of hinds. We used three experimental groups, including the ovaries collected post mortem on the 4th and 13th days of the estrous cycle and during pregnancy (n = 18). After oocyte maturation, frozen-thawed epididymal semen was used for IVF. Blastocyst quality, apoptotic potential by determining the mRNA expression of BAX, BCL-2, OCT4, SOX2, and placenta-specific 8 gene (PLAC8), and antioxidative potential of blastocysts were evaluated by determining the mRNA expression of CuSOD, MnSOD, and GPX as well as the enzymatic activity of superoxide dismutase and reduced glutathione. The highest development rate of expanded blastocyst, mRNA expression of BCL-2, OCT4, SOX2, and PLAC8 and mRNA expression and enzymatic activity of the antioxidative factors increased (p < 0.05) in blastocysts developed from the oocytes collected on the 4th day, compared to those developed from the oocytes collected on the 13th day of the cycle and during pregnancy. Our study indicates that the 4th day of the estrous cycle is the most effective period for oocyte collection for IVF and embryo development in hinds, considering quality parameters and antioxidative potential of the blastocysts.

Profiles of maternal origin factors during transition from embryonic diapause to implantation in roe deer.

The aim was to evaluate in female roe deer: (a) PAG mRNA relative abundance in endometrial uterine tissue for determination of the duration of embryonic diapause, (b) mRNA relative abundance of progesterone, estradiol, and prolactin (P4, E2, and PRL) receptors (PGR, ESR, and PRLR) during diapause and after implantation in the endometrium; (c) concentration of P4, E2, and PRL in the blood, and (d) a noninvasive method of hormone detection by measurement of P4 and E2 concentrations in feces. A total of fifteen individuals were obtained post mortem during hunting seasons and divided into three experimental groups (November, December, January). The results did not reveal mRNA relative abundance for PAGs in the endometrium or detectable PAG concentrations in the serum of all examined females. Concentration of PRL and mRNA relative abundance for PRLR long isoform in the endometrium was the highest in January (p < .01). mRNA relative abundance for PGR, P4 concentration in the endometrium, serum, and feces was the highest in January (p < .01). Endometrial origin PRL and P4 may be responsible for the termination of this process and pregnancy development after implantation.